CN114752588A - Heparinase II - Google Patents
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Abstract
The application discloses heparinase II, a coding nucleotide sequence thereof, a recombinant vector and a host cell comprising the nucleotide sequence and application thereof, wherein the heparinase II carries out site-directed mutagenesis on the amino acid sequence of the existing heparinase II, specifically, glutamine (Q) at three sites is subjected to site-directed mutagenesis to alanine (A) or valine (V), and the heparinase II obtained after the mutagenesis has better stability compared with the existing heparinase II under the condition of not influencing the activity of the heparinase II.
Description
Technical Field
The present invention relates generally to the field of biological genetic engineering and fermentation engineering. Within this field, the present invention relates to heparinase II and its encoding gene, and further provides a method for preparing heparinase II using a recombinant vector and a host cell.
Background
Heparinases (heparinases) are a kind of polysaccharide lyases acting on heparin or heparan sulfate, are used for researching the interaction between heparinases and a substrate thereof, namely polysaccharide heparin, are helpful for explaining the action mechanism of the polysaccharide lyases, and have important application in various aspects such as analyzing the structure and biological functions of complex mucopolysaccharides such as heparin, analyzing the coagulation and anticoagulation mechanisms in human bodies, and only being low-molecular anticoagulant drugs such as anticoagulant drugs of low molecular weight and clinical application. Heparinases are found in many microorganisms, of which there are three major heparinases from Flavobacterium heparinum, heparinase II (EC4.2.2.7), heparinase II (No EC code) and heparinase III (EC4.2.2.8), respectively.
The natural heparinase II is usually obtained by purifying from a Flavobacterium heparinum fermentation liquid, the heparinase II is produced by the Flavobacterium heparinum while the heparinase I, III and four chondroitinases (chondroitinase B, C, ABC and AC) are produced, so that the separation and purification of the heparinase II are complicated, multi-step chromatographic purification is usually required, the activity loss of the heparinase is extremely large, the yield is low, and an inducing additive heparin sodium of the heparinase II can only be extracted from small intestinal mucosa of animals (mainly pigs and cattle), the process is complex, the cost is high, and the yield and the application of the heparinase II are severely limited.
Disclosure of Invention
The stability of the heparinase II prepared by the existing method is poor, the activity of the heparinase II prepared by the existing method is reduced to half of the activity of the heparinase II prepared by the existing method when the heparinase II is stored at low temperature in a short time, and the activity of the heparinase II can be seriously lost after one-time freeze thawing and one-time freeze drying. Therefore, there is a need in the art for improvements in the current heparinase II and methods of making the same.
In view of the above, the inventors of the present application have made intensive studies and have made the present invention.
Compared with the original heparinase II, the heparinase II provided by the invention has stronger stability under the condition of not influencing enzyme activity, and the invention also provides a method for preparing the heparinase II.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a heparinase II comprising a sequence as set forth in SEQ ID NO: 02, the amino acid sequence is obtained by site-directed mutagenesis of glutamine (Q) at a plurality of positions of the amino acid sequence of the original heparinase II (shown as SEQ ID NO: 01) to alanine (A), and meanwhile, in order to improve the protein purification efficiency, a strepII tag sequence is added at the C end of the heparinase II, and the cell crude extract can pass through a desulfurization biotin purification column, and the purification of the target heparinase II is realized by the interaction between the strepII tag and biotin.
The invention also provides a nucleotide sequence for coding the heparinase II.
Preferably, the nucleotide sequence is as shown in SEQ ID NO: 04, respectively.
As a preferred embodiment, the present invention also provides a heparinase II comprising a sequence as set forth in SEQ ID NO: 03, the amino acid sequence is obtained by site-directed mutagenesis of glutamine (Q) at multiple positions of the amino acid sequence of the original heparinase II (shown as SEQ ID NO: 01) into alanine (A) or valine (V), and meanwhile, in order to improve the protein purification efficiency, a strepII tag sequence is added at the C end of the heparinase II, and the cell crude extract can pass through a desulfurization biotin purification column and realize the purification of the target heparinase II by virtue of the interaction between the strepII tag and biotin.
The invention also provides a method for coding the amino acid sequence shown as SEQ ID NO: 03, and a heparinase II nucleotide sequence.
Preferably, the nucleotide sequence is as shown in SEQ ID NO: 05, and (b).
In another aspect, the present invention provides a recombinant vector comprising the above nucleotide sequence.
Further, the recombinant vector comprises a eukaryotic cell recombinant expression vector.
Furthermore, the eukaryotic cell recombinant expression vector comprises any one of pPink-HC, pPICZaA and pPICZ A;
as a preferred method, the eukaryotic cell recombinant expression vector is pPink-HC.
The invention also provides a host cell which comprises the recombinant vector.
Further, the host cell is one of pichia or saccharomyces cerevisiae.
Further, the host cell is pichia pastoris.
In one aspect, the invention provides a method for preparing the heparinase II, comprising the steps of:
firstly, synthesizing a nucleotide sequence of the heparinase II, and then combining the nucleotide sequence with a eukaryotic cell recombinant expression vector to obtain a recombinant vector;
And transferring the recombinant vector into a host cell, then inducing expression, and purifying to obtain the heparinase II.
Preferably, the host cell is one of pPink-HC, pPICZaA and pPICZA, and in a further preferred scheme, the eukaryotic cell recombinant expression vector is pPink-HC.
Preferably, in the above preparation method, the step of combining the nucleotide sequence with the eukaryotic recombinant expression vector and the recombinant vector is performed according to the instructions of the Pichia Pink system kit.
Preferably, in the above preparation method, the step of inducing expression comprises: transforming yeast, peptone and YNB into the recombinant expression vector, adding water and phosphate buffer, subpackaging BMMY, adding glycerol into the rest culture medium, subpackaging BMGY, selecting positive transformants, inoculating into a shake flask of the BMGY culture medium, culturing, centrifuging to remove supernatant, taking BMMY to resuspend thalli, adding into a shake flask filled with 20-25mL BMMY culture medium, controlling the initial OD600 to be about 1, continuing culturing, regularly sampling and adding methanol, measuring the OD600 and the foreign protein expression amount, and after fermentation is completed, centrifugally collecting fermentation liquor.
Preferably, in the above preparation method, the purification step comprises: collecting thalli (3-6 ℃), using 1-2 ml Buffer W (precooling at 3-6 ℃) suspension for every 100ml of collected thalli, adding a protease inhibitor, and breaking cells on an ice water mixture by ultrasound to obtain a lysate. And then purifying, specifically, washing a purification column by using Buffer W, slowly loading lysis solution (3-6 ℃) of 0.5-10CVs into the column, washing the column by using Buffer W after the sample completely enters the column, collecting eluent of each part, adding 4-7 times of Buffer E, collecting in each section, and operating in a low-temperature chromatography cabinet in the whole process.
Further, the purification column is a desthiobiotin purification column.
Compared with the prior art, the invention has the beneficial effects that:
the heparinase II provided by the invention comprises a heparinase II shown as SEQ ID NO: 02 or as shown in SEQ ID NO: 03. The invention mutates protease enzyme cutting sites which may affect the stability of the heparinase II in the amino acid sequence (shown as SEQ ID NO: 01) of the original heparinase II, and concretely, the glutamine (Q) at a plurality of positions of the amino acid sequence of the natural heparinase II (shown as SEQ ID NO: 01) is mutated into alanine (A) or valine (V) at fixed points. The enzyme activity of the heparinase II obtained after point mutation is not obviously reduced, and the enzyme activity stability of the two heparinases II is obviously improved compared with that of the original heparinases II under the condition of 30 ℃.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts. Wherein:
FIG. 1 shows the result of the identification of purified electrophoresis provided in example 4 of the present invention;
FIG. 2 shows the enzyme activity stability analysis provided in example 6 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The original heparinase II has an amino acid sequence shown as SEQ ID NO: 01, from the published sequence of heparinase II in the NCBI database, the NCBI database website is: https:// www.ncbi.nlm.nih.gov/protein/ACB 38160.1.
Heparinase II cleaves heparin sulfate and heparin mainly at 1-4 connecting sites of hexosamine and uronic acid (glucuronic acid and iduronic acid), the product is mainly disaccharide, which has good degradation effect on heparin substances, but the stability of original heparinase II is poor, generally, the purified heparinase II needs multi-step operation, in the process, the loss of enzyme activity is huge, and the application of the heparinase II is limited due to the low yield of less than 10 percent. Although certain substances can be used in the purification process to improve the service life of the enzyme, further research on the structure and catalytic mechanism of the enzyme is needed to fundamentally solve the problem of instability of the enzyme.
The invention provides a heparinase II, which is obtained by site-directed mutating glutamine (Q) at 77, 261 and 276 sites in an amino acid sequence of an original heparinase II into alanine (A) or valine (V) to obtain the heparinase II which comprises a nucleotide sequence shown in SEQ ID NO: 02 or SEQ ID NO: 03, and a pharmaceutically acceptable salt thereof.
According to the reported crystal structure sequence information of heparinase II, 77, 261 and 276 sites in the original heparinase II amino acid sequence are located in a protein outer negative charge accumulation region in the heparinase II structure and weak positions in the structure, when the heparinase II is expressed, the heparinase II can be attacked by protease to be degraded, particularly, in the purification process of the heparinase, a large amount of protease is released after thalli are cracked, and the heparinase II is extremely easy to degrade to influence the stability of the heparinase II. Therefore, the invention respectively carries out amino acid substitution and compound mutation on the three sites of 77, 261 and 276, particularly, the site-specific mutation of glutamine (Q) of the three sites into alanine (A) or valine (V) can obviously improve the enzyme activity stability of heparinase II. Meanwhile, activity determination proves that mutation modification of the site does not affect the activity of the heparinase II and has better stability compared with the original heparinase II.
Wherein, the site-specific mutagenesis of 77 and 261 glutamine (Q) in the amino acid sequence of the original heparinase II to alanine (A) is carried out, and the heparinase II obtained by the site-specific mutagenesis of 276 glutamine (Q) to valine (V) is stronger in enzyme activity stability than the heparinase II obtained by the site-specific mutagenesis of three sites of glutamine (Q) to alanine (A), and the activity of the heparinase II is not obviously reduced compared with that of the original heparinase II.
The invention provides a nucleotide sequence for coding the heparinase II, wherein the nucleotide sequence for coding the heparinase II is any one nucleotide sequence capable of expressing the heparinase II, and as a preferred embodiment, the nucleotide sequence is shown as SEQ ID NO: 04 or SEQ ID NO: 05, and (b).
The invention provides a recombinant vector, which comprises any one of the nucleotide sequences in copy and is used for expressing heparinase II.
The recombinant vector refers to a DNA sequence which can be inserted with an exogenous DNA sequence and can be autonomously replicated, and a consciously modified amino acid sequence or nucleotide sequence, and generally comprises a prokaryotic expression vector and a eukaryotic cell recombinant expression vector.
The eukaryotic cell recombinant expression vector can produce glycosylated soluble expression heparinase II, while the heparinase II expressed by the prokaryotic expression vector cannot carry out glycosylation on protein, most of the protein is in an inactive inclusion body form, and the active heparinase II cannot be obtained by purification. Therefore, compared with a prokaryotic expression vector, the heparinase II obtained by the eukaryotic cell recombinant expression vector has higher activity. The species include pPIC9K, pPICZaA, pGAPZaA, pPIC3.5K, pPink-HC, pPICZaA, pPICZA, etc.
In a preferred embodiment, the eukaryotic cell recombinant expression vector comprises one of pPink-HC, pPICZaA and pPICZA, and in a further preferred embodiment, the eukaryotic cell recombinant expression vector is pPink-HC.
The invention provides a host cell, which comprises the recombinant vector and is used for expressing heparinase II.
Such host cells include unicellular prokaryotic and eukaryotic organisms (e.g., bacteria, yeast and actinomycetes) and unicellular cells from higher plants or animals when grown in cell culture, Flavobacterium heparinum is a common host cell for expression of heparinase II, and in recent years, Escherichia coli and yeast, among others, have also been used for expression of heparinase II.
The host cell provided by the invention can be any host cell for expressing the heparinase II, and as a preferred embodiment of the invention, the host cell is one of pichia or saccharomyces cerevisiae, and preferably, the host cell is pichia.
The pichia pastoris is used as a host cell to express the foreign protein, is a novel high-efficiency expression system, contains a special strong alcohol oxidase gene promoter, can strictly regulate and control the expression of the foreign gene by using methanol, has stable genetic inheritance of the foreign protein gene, is integrated into a pichia pastoris genome with high copy number, is not easy to lose and can obtain a high-expression strain, and has a subcellular structure of eukaryote and post-translational modification processing functions of glycosylation, fatty acylation, protein phosphorylation and the like. The culture cost of the pichia pastoris is very low, the used fermentation medium is very cheap, the common carbon sources are glycerol or glucose and methanol, the rest is inorganic salt, and the culture medium does not contain protein, so that the separation and purification of downstream products are facilitated, and the products are easy to separate.
The invention provides a preparation method of heparinase II, which comprises the following steps: firstly, synthesizing a nucleotide sequence for encoding heparinase II related to the text, and then combining the nucleotide sequence with a eukaryotic cell recombinant expression vector to obtain a recombinant vector; and transferring the recombinant vector into a host cell, inducing expression, and purifying to obtain the heparinase II.
In a preferred embodiment of the present invention, in the above preparation method, the step of synthesizing a nucleotide sequence encoding heparinase II referred to herein is as follows:
firstly, the amino acid sequence of the original heparinase II is reversely translated into a nucleotide sequence, preferably, the obtained nucleotide sequence is preferred by a selected host cell.
In a preferred embodiment of the present invention, the nucleotide sequence of heparinase II is combined with a eukaryotic cell recombinant expression vector to obtain a recombinant vector, and the steps of:
firstly, enzyme cutting sites of connection points on the recombinant expression vector are cut off, and then heparinase II nucleotide is connected with the cut-off expression vector to construct the recombinant expression vector.
In a preferred embodiment of the present invention, in the above preparation method, the host cell is one of pPink-HC, pPICZaA and pPICZA, and in a further preferred embodiment, the eukaryotic cell recombinant expression vector is pPink-HC.
In a preferred embodiment of the present invention, in the above preparation method, the step of combining the nucleotide sequence with the eukaryotic recombinant expression vector and the recombinant vector is performed according to the instructions of the Pichia Pink system kit.
In a preferred embodiment of the present invention, in the above preparation method, the step of inducing expression is as follows:
transforming yeast, peptone and YNB by using the recombinant expression vector, adding water and phosphate buffer solution, subpackaging BMMY, adding glycerol into the rest culture medium, then subpackaging BMGY, selecting a positive transformant, inoculating the positive transformant into a shake flask of the BMGY culture medium, culturing, centrifuging to remove supernatant, taking BMMY to resuspend thalli, adding the bmMY into the shake flask filled with 20-25mL of BMMY culture medium, controlling the initial OD600 to be about 1, continuing to culture, regularly sampling and adding methanol, measuring the OD600 and the expression quantity of an exogenous protein, and after fermentation is completed, centrifuging and collecting fermentation liquor.
The YNB culture medium is also called as an amino-free yeast nitrogen culture medium and is used for fermentation culture of yeast.
The BMMY culture medium is a seed liquid culture medium, namely an induction expression culture medium, and is used for methanol induction of pichia pastoris recombinant strains to secrete and express target proteins.
The peptone is faint yellow powder which is prepared by hydrolyzing meat, casein or gelatin by acid or protease and then drying, and provides a required carbon source for yeast cells.
In a preferred embodiment of the present invention, in the above preparation method, the purification steps are as follows:
Collecting thalli (3-6 ℃), using 1-2 ml Buffer W (precooling at 3-6 ℃) suspension for every 100ml of collected thalli, adding a protease inhibitor, and breaking cells on an ice water mixture by ultrasound to obtain a lysate. And then purifying, specifically, washing a purification column by using Buffer W, slowly loading lysis solution (3-6 ℃) of 0.5-10CVs into the column, washing the column by using Buffer W after the sample completely enters the column, collecting eluent of each part, adding 4-7 times of Buffer E, collecting in each section, and operating in a low-temperature chromatography cabinet in the whole process.
The wash Buffer W (wash Buffer) rinsing liquid is used for washing off the hybrid protein specifically combined with the purification column waste
The Buffer E (elution Buffer) eluent has the function of eluting the target protein specifically bound with the purification column, namely heparinase.
As a preferable scheme of the invention, the purification column is a desthiobiotin purification column, in order to improve the protein purification efficiency, a strepII tag sequence is added at the C end of the heparinase II, and the interaction between the strepII tag and biotin in the desthiobiotin purification column is utilized to realize better purification effect of the target protein heparinase II.
Has the beneficial effects that:
under the condition of not influencing enzyme activity, compared with the original heparinase II, the heparinase II provided by the invention has the advantages that the enzyme activity stability under the condition of 30 ℃ is obviously improved, the enzyme activity half-life period is improved by nearly one time, and the stability is stronger.
The following examples of the present invention are merely illustrative of specific embodiments for carrying out the present invention and should not be construed as limiting the invention. Other changes, modifications, substitutions, combinations, and simplifications which may be made without departing from the spirit and principles of the present invention are intended to be equivalents thereof and are intended to be included within the scope of the present invention.
Examples
Materials, reagents and the like used in the following examples were commercially available unless otherwise specified.
Experimental materials used in Table 1
| Name of raw materials | Type/purity | Manufacturer of the product |
| Pichia Pink system kit | A11152 | Thermo Fisher Co Ltd |
| YNB medium | Y8040-100g | Solarbio |
| Peptone | LP0042 | OXOID |
| Strep-Tactin column (cysteine-containing column) | BTR211Q | Boersi (Borneo) |
| Heparin sodium | NHS200803 | Hebei Changshan Biochemical pharmaceutical Co., Ltd |
Example 1 improvement of the optimization of the nucleotide sequence of heparinase II and construction of an expression vector
(a) The amino acid sequence of the original heparinase II is a sequence from heparinase II published in the NCBI database with the website: https:// www.ncbi.nlm.nih.gov/protein/ACB 38160.1.
(b) The protein sequence was reverse translated into a DNA sequence by Cincirus Soviensis Biotech, Suzhou according to the codon usage bias of Pichia pastoris in the Pichia pastoris codon bias data sheet, so that the codons of the DNA sequence were all Pichia pastoris biased.
(c) Carrying out mutation modification on potential protease cleavage sites, specifically, replacing glutamine (Q) at the amino acid sequences 77, 261 and 276 of an original heparinase II with alanine (A) to obtain heparinase II-1, replacing the amino acid sequences 77 and 261 of the original heparinase II with alanine (A), replacing the glutamine (Q) at the 276 of the original heparinase II with valine (V) to obtain heparinase II-2, and respectively carrying out full-sequence synthesis on the two modified sequences of heparinase II-1 and heparinase II-2 to obtain a heparinase II nucleotide sequence.
(d) And (3) selecting EcoRI and EcoRV enzyme cutting sites on the pPink-HC recombinant expression vector to disconnect the expression vector, connecting heparinase II with the disconnected expression vector, and constructing the HepI-pPink-HC recombinant expression vector.
Example 2 competent preparation and electrotransformation
The procedure was followed according to the instructions for the Pichia Pink system kit.
Example 3 inducible expression of heparinase II
Firstly, preparing a culture medium:
(1) BMGY liquid Medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB, 1% (w/v) glycerol, 10% (v/v)1M phosphate buffer pH 6.0. Sterilizing at 115 deg.C for 20 min;
(2) BMMY liquid medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB, 1% (v/v) methanol, 10% (v/v)1M phosphate buffer pH 6.0. Sterilizing at 115 deg.C for 20 min.
II, inducing expression: weighing yeast extract, peptone and YNB, adding water and phosphate buffer, packaging BMMY, adding glycerol to the rest culture medium, and packaging BMGY.
Thirdly, fermentation process:
three positive transformants were selected and inoculated into 50mL shake flasks containing 5-10mL BMGY medium, incubated at 30 ℃ and 250rpm until OD600 (optical density at 600 nm) was 4(16-18 hours), centrifuged at 3000g for 3min to remove the supernatant, 1mL BMMY-resuspended cells were added to 250mL shake flasks containing 23mL BMMY medium with the initial OD600 controlled at about 1, incubated at 30 ℃ and 250rpm, sampled (-200 uL) and added with 1% methanol every 24 hours to measure OD600 and foreign protein expression, fermentation was stopped after 96 hours, and the fermentation broth was collected by centrifugation.
Example 4 purification
First, treatment of cell disruption lysate before purification
Proportioning of Buffer W solution: 20mM Na2HPO4, 0.28M NaCl, 6mM KCl, pH 7.4.
Proportioning of Buffer E solution: 20mM Na2HPO4, 0.28M NaCl, 6mM KCl, 2.5mM desthiobiotin, pH 7.4.
The cells were collected (4500g, 15min, 4 ℃ C.), suspended in 1ml Buffer W (precooled at 4 ℃ C.) per 100ml of the collected cells, added with a protease inhibitor, and disrupted by ultrasonication on an ice-water mixture to obtain a lysate.
Secondly, purification and identification
Washing the Strep-Tactin column (cysteine-containing column) with Buffer W of 2CVs (binding material of column is cysteine, elution is desthiobiotin), slowly loading the lysate of 5CVs (4 ℃) on the column, after the sample completely enters the column, washing the column with Buffer W of 5CVs, collecting the eluate of each fraction, adding Buffer E of 0.5CVs 6 times, and collecting at each fraction (0.5 CVs). Each fraction was collected and identified by 20. mu.l SDS-PAGE, the fusion tag protein was usually at 2ndAnd 5thAnd (4) part (a). The whole process is operated in a low-temperature chromatography cabinet at 4 ℃.
The result of SDS-PAGE electrophoretic analysis is shown in figure 1, which shows that the method can successfully obtain the heparinase II protein with the purity of more than 90 percent, and the molecular weight is consistent with the expected molecular weight.
EXAMPLE 5 protein Activity assay
The substrate was heparin sodium (Hebei dichroa biochemical pharmaceuticals, Inc.) and the absorbance was measured as a time-dependent change curve using an ultraviolet-visible spectrophotometer (GOLDS 54, Shanghai Ling light technology, Inc.). The scanning wavelength was 232nm and the time was 3 min. Taking reaction buffer (20mM Tris, 200mM NaCl, fully dissolved, then adjusting pH to 7.4 with 6M hydrochloric acid, preserving at 4 ℃), about 1000 μ L with a certain amount of enzyme solution (the specific proportion needs to be adjusted according to the activity of the enzyme solution, generally 1ml buffer, 2 μ L enzyme solution), 500 μ L substrate solution (17mM Tris, 44mM NaCl, 3.5mM CaCl2, 25g/L sodium heparin, fully stirring, then adjusting pH to 7.0 with 6M hydrochloric acid, preserving at 4 ℃) in a quartz cuvette, immediately placing the cuvette into a spectrophotometer for scanning after mixing (the reaction buffer and the substrate solution are preheated to constant temperature in a 30 ℃ water bath for at least 30min before mixing), scanning for 70s heparin, taking data of 40-60s, calculating the slope k (min-1) after finishing, and then calculating the enzyme activity (IU/L) of the enzyme and deducing the process are as follows:
According to beer's law, the absorbance a ═ ε c, where ε is 3800M-1.cm-1, so that the total activity of the enzyme in the 1500 μ L reaction system was 15/38k (min-1) IU, and if the volume of the enzyme solution added to the 1500 μ L reaction system was V (μ L), the enzyme activity of the added enzyme solution was calculated as follows:
the 3 mutation sites selected by the invention are 77, 261 and 276 respectively, and are subjected to amino acid substitution and compound mutation one by one, and the data of enzyme activity and stability are tested. In the structure of heparinase II, the 3 sites are all located in the protein outer negative charge aggregation region and the weaker position in the structure, and the enzyme activity and the stability are probably influenced greatly.
Under the same conditions of fermentation, crushing and purification, the enzyme activity data of the mutant are shown in table 2, and as can be seen from table 2, the enzyme activity of the mutant of Q77A is reduced to a certain extent relative to that of the original heparinase II; the enzyme activity of the mutants of Q261A, Q276A, Q261A and Q276A is slightly reduced, wherein the enzyme activity of the mutants of heparinase II-1 and heparinase is closest to that of the original heparinase I, and the reduction is least.
Table 2: results of enzyme Activity measurement
| Mutation site | Enzyme activity (IU/L) |
| Not mutated | 882.02 |
| Q77A | 655.12 |
| Q261A | 1133.95 |
| Q276A | 1032.55 |
| Q261A&Q276A | 1311.54 |
Example 6 thermal stability analysis
And respectively placing the purified heparinase II-1, the purified heparinase II-2 and the heparinase II with the original sequence on ice, immediately detecting the enzyme activity, recording the time as 0, and taking the enzyme activity value as 100%. And then, placing the enzyme in a warm bath at 30 ℃, sampling every 10min to determine the enzyme activity, and recording the ratio of the enzyme activity to the enzyme activity value at 0 moment. The detection time is stopped until one of the heparinases II reaches half-life. When the stability of different enzymes is compared, the determination is made based on the inactivation rates of the enzymes at the same concentration, under the same solution conditions, and under the same temperature bath conditions. (enzyme activity was measured 3 times in parallel and averaged)
The analysis result is shown in fig. 2, and it can be seen from fig. 2 that, under the same temperature bath condition of 30 ℃, the thermal stability of the improved heparinase II is remarkably improved compared with that of the heparinase II of the original sequence, the half-life of the enzyme activity of the heparinase II-1 is improved from about 11h to about 50h, and the stability of the heparinase II-2 is further improved, which indicates that the 276 is more critical for the heparinase II to resist the intracellular protease, and the 276 is more beneficial to the improvement of the stability of the heparinase II when the position is valine.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Sequence listing
<110> International Tech of Edehake in Beijing
<120> a heparinase II
<130> TPE01496
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 756
<212> PRT
<213> Artificial sequence
<220>
<223> description of artificial sequences: artificially synthesized sequence
<400> 1
Met Gln Thr Lys Ala Asp Val Val Trp Lys Asp Val Asp Gly Val Ser
1 5 10 15
Met Pro Ile Pro Pro Lys Thr His Pro Arg Leu Tyr Leu Arg Glu Gln
20 25 30
Gln Val Pro Asp Leu Lys Asn Arg Met Asn Asp Pro Lys Leu Lys Lys
35 40 45
Val Trp Ala Asp Met Ile Lys Met Gln Glu Asp Trp Lys Pro Ala Asp
50 55 60
Ile Pro Glu Val Lys Asp Phe Arg Phe Tyr Phe Asn Gln Lys Gly Leu
65 70 75 80
Thr Val Arg Val Glu Leu Met Ala Leu Asn Tyr Leu Met Thr Lys Asp
85 90 95
Pro Lys Val Gly Arg Glu Ala Ile Thr Ser Ile Ile Asp Thr Leu Glu
100 105 110
Thr Ala Thr Phe Lys Pro Ala Gly Asp Ile Ser Arg Gly Ile Gly Leu
115 120 125
Phe Met Val Thr Gly Ala Ile Val Tyr Asp Trp Cys Tyr Asp Gln Leu
130 135 140
Lys Pro Glu Glu Lys Thr Arg Phe Val Lys Ala Phe Val Arg Leu Ala
145 150 155 160
Lys Met Leu Glu Cys Gly Tyr Pro Pro Val Lys Asp Lys Ser Ile Val
165 170 175
Gly His Ala Ser Glu Trp Met Ile Met Arg Asp Leu Leu Ser Val Gly
180 185 190
Ile Ala Ile Tyr Asp Glu Phe Pro Glu Met Tyr Asn Leu Ala Ala Gly
195 200 205
Arg Phe Phe Lys Glu His Leu Val Ala Arg Asn Trp Phe Tyr Pro Ser
210 215 220
His Asn Tyr His Gln Gly Met Ser Tyr Leu Asn Val Arg Phe Thr Asn
225 230 235 240
Asp Leu Phe Ala Leu Trp Ile Leu Asp Arg Met Gly Ala Gly Asn Val
245 250 255
Phe Asn Pro Gly Gln Gln Phe Ile Leu Tyr Asp Ala Ile Tyr Lys Arg
260 265 270
Arg Pro Asp Gly Gln Ile Leu Ala Gly Gly Asp Val Asp Tyr Ser Arg
275 280 285
Lys Lys Pro Lys Tyr Tyr Thr Met Pro Ala Leu Leu Ala Gly Ser Tyr
290 295 300
Tyr Lys Asp Glu Tyr Leu Asn Tyr Glu Phe Leu Lys Asp Pro Asn Val
305 310 315 320
Glu Pro His Cys Lys Leu Phe Glu Phe Leu Trp Arg Asp Thr Gln Leu
325 330 335
Gly Ser Arg Lys Pro Asp Asp Leu Pro Leu Ser Arg Tyr Ser Gly Ser
340 345 350
Pro Phe Gly Trp Met Ile Ala Arg Thr Gly Trp Gly Pro Glu Ser Val
355 360 365
Ile Ala Glu Met Lys Val Asn Glu Tyr Ser Phe Leu Asn His Gln His
370 375 380
Gln Asp Ala Gly Ala Phe Gln Ile Tyr Tyr Lys Gly Pro Leu Ala Ile
385 390 395 400
Asp Ala Gly Ser Tyr Thr Gly Ser Ser Gly Gly Tyr Asn Ser Pro His
405 410 415
Asn Lys Asn Phe Phe Lys Arg Thr Ile Ala His Asn Ser Leu Leu Ile
420 425 430
Tyr Asp Pro Lys Glu Thr Phe Ser Ser Ser Gly Tyr Gly Gly Ser Asp
435 440 445
His Thr Asp Phe Ala Ala Asn Asp Gly Gly Gln Arg Leu Pro Gly Lys
450 455 460
Gly Trp Ile Ala Pro Arg Asp Leu Lys Glu Met Leu Ala Gly Asp Phe
465 470 475 480
Arg Thr Gly Lys Ile Leu Ala Gln Gly Phe Gly Pro Asp Asn Gln Thr
485 490 495
Pro Asp Tyr Thr Tyr Leu Lys Gly Asp Ile Thr Ala Ala Tyr Ser Ala
500 505 510
Lys Val Lys Glu Val Lys Arg Ser Phe Leu Phe Leu Asn Leu Lys Asp
515 520 525
Ala Lys Val Pro Ala Ala Met Ile Val Phe Asp Lys Val Val Ala Ser
530 535 540
Asn Pro Asp Phe Lys Lys Phe Trp Leu Leu His Ser Ile Glu Gln Pro
545 550 555 560
Glu Ile Lys Gly Asn Gln Ile Thr Ile Lys Arg Thr Lys Asn Gly Asp
565 570 575
Ser Gly Met Leu Val Asn Thr Ala Leu Leu Pro Asp Ala Ala Asn Ser
580 585 590
Asn Ile Thr Ser Ile Gly Gly Lys Gly Lys Asp Phe Trp Val Phe Gly
595 600 605
Thr Asn Tyr Thr Asn Asp Pro Lys Pro Gly Thr Asp Glu Ala Leu Glu
610 615 620
Arg Gly Glu Trp Arg Val Glu Ile Thr Pro Lys Lys Ala Ala Ala Glu
625 630 635 640
Asp Tyr Tyr Leu Asn Val Ile Gln Ile Ala Asp Asn Thr Gln Gln Lys
645 650 655
Leu His Glu Val Lys Arg Ile Asp Gly Asp Lys Val Val Gly Val Gln
660 665 670
Leu Ala Asp Arg Ile Val Thr Phe Ser Lys Thr Ser Glu Thr Val Asp
675 680 685
Arg Pro Phe Gly Phe Ser Val Val Gly Lys Gly Thr Phe Lys Phe Val
690 695 700
Met Thr Asp Leu Leu Pro Gly Thr Trp Gln Val Leu Lys Asp Gly Lys
705 710 715 720
Ile Leu Tyr Pro Ala Leu Ser Ala Lys Gly Asp Asp Gly Pro Leu Tyr
725 730 735
Phe Glu Gly Thr Glu Gly Thr Tyr Arg Phe Leu Arg Trp Ser His Pro
740 745 750
Gln Phe Glu Lys
755
<210> 2
<211> 756
<212> PRT
<213> Artificial sequence
<220>
<223> description of artificial sequences: artificially synthesized sequence
<400> 2
Met Gln Thr Lys Ala Asp Val Val Trp Lys Asp Val Asp Gly Val Ser
1 5 10 15
Met Pro Ile Pro Pro Lys Thr His Pro Arg Leu Tyr Leu Arg Glu Gln
20 25 30
Gln Val Pro Asp Leu Lys Asn Arg Met Asn Asp Pro Lys Leu Lys Lys
35 40 45
Val Trp Ala Asp Met Ile Lys Met Gln Glu Asp Trp Lys Pro Ala Asp
50 55 60
Ile Pro Glu Val Lys Asp Phe Arg Phe Tyr Phe Asn Ala Lys Gly Leu
65 70 75 80
Thr Val Arg Val Glu Leu Met Ala Leu Asn Tyr Leu Met Thr Lys Asp
85 90 95
Pro Lys Val Gly Arg Glu Ala Ile Thr Ser Ile Ile Asp Thr Leu Glu
100 105 110
Thr Ala Thr Phe Lys Pro Ala Gly Asp Ile Ser Arg Gly Ile Gly Leu
115 120 125
Phe Met Val Thr Gly Ala Ile Val Tyr Asp Trp Cys Tyr Asp Gln Leu
130 135 140
Lys Pro Glu Glu Lys Thr Arg Phe Val Lys Ala Phe Val Arg Leu Ala
145 150 155 160
Lys Met Leu Glu Cys Gly Tyr Pro Pro Val Lys Asp Lys Ser Ile Val
165 170 175
Gly His Ala Ser Glu Trp Met Ile Met Arg Asp Leu Leu Ser Val Gly
180 185 190
Ile Ala Ile Tyr Asp Glu Phe Pro Glu Met Tyr Asn Leu Ala Ala Gly
195 200 205
Arg Phe Phe Lys Glu His Leu Val Ala Arg Asn Trp Phe Tyr Pro Ser
210 215 220
His Asn Tyr His Gln Gly Met Ser Tyr Leu Asn Val Arg Phe Thr Asn
225 230 235 240
Asp Leu Phe Ala Leu Trp Ile Leu Asp Arg Met Gly Ala Gly Asn Val
245 250 255
Phe Asn Pro Gly Gln Ala Phe Ile Leu Tyr Asp Ala Ile Tyr Lys Arg
260 265 270
Arg Pro Asp Gly Ala Ile Leu Ala Gly Gly Asp Val Asp Tyr Ser Arg
275 280 285
Lys Lys Pro Lys Tyr Tyr Thr Met Pro Ala Leu Leu Ala Gly Ser Tyr
290 295 300
Tyr Lys Asp Glu Tyr Leu Asn Tyr Glu Phe Leu Lys Asp Pro Asn Val
305 310 315 320
Glu Pro His Cys Lys Leu Phe Glu Phe Leu Trp Arg Asp Thr Gln Leu
325 330 335
Gly Ser Arg Lys Pro Asp Asp Leu Pro Leu Ser Arg Tyr Ser Gly Ser
340 345 350
Pro Phe Gly Trp Met Ile Ala Arg Thr Gly Trp Gly Pro Glu Ser Val
355 360 365
Ile Ala Glu Met Lys Val Asn Glu Tyr Ser Phe Leu Asn His Gln His
370 375 380
Gln Asp Ala Gly Ala Phe Gln Ile Tyr Tyr Lys Gly Pro Leu Ala Ile
385 390 395 400
Asp Ala Gly Ser Tyr Thr Gly Ser Ser Gly Gly Tyr Asn Ser Pro His
405 410 415
Asn Lys Asn Phe Phe Lys Arg Thr Ile Ala His Asn Ser Leu Leu Ile
420 425 430
Tyr Asp Pro Lys Glu Thr Phe Ser Ser Ser Gly Tyr Gly Gly Ser Asp
435 440 445
His Thr Asp Phe Ala Ala Asn Asp Gly Gly Gln Arg Leu Pro Gly Lys
450 455 460
Gly Trp Ile Ala Pro Arg Asp Leu Lys Glu Met Leu Ala Gly Asp Phe
465 470 475 480
Arg Thr Gly Lys Ile Leu Ala Gln Gly Phe Gly Pro Asp Asn Gln Thr
485 490 495
Pro Asp Tyr Thr Tyr Leu Lys Gly Asp Ile Thr Ala Ala Tyr Ser Ala
500 505 510
Lys Val Lys Glu Val Lys Arg Ser Phe Leu Phe Leu Asn Leu Lys Asp
515 520 525
Ala Lys Val Pro Ala Ala Met Ile Val Phe Asp Lys Val Val Ala Ser
530 535 540
Asn Pro Asp Phe Lys Lys Phe Trp Leu Leu His Ser Ile Glu Gln Pro
545 550 555 560
Glu Ile Lys Gly Asn Gln Ile Thr Ile Lys Arg Thr Lys Asn Gly Asp
565 570 575
Ser Gly Met Leu Val Asn Thr Ala Leu Leu Pro Asp Ala Ala Asn Ser
580 585 590
Asn Ile Thr Ser Ile Gly Gly Lys Gly Lys Asp Phe Trp Val Phe Gly
595 600 605
Thr Asn Tyr Thr Asn Asp Pro Lys Pro Gly Thr Asp Glu Ala Leu Glu
610 615 620
Arg Gly Glu Trp Arg Val Glu Ile Thr Pro Lys Lys Ala Ala Ala Glu
625 630 635 640
Asp Tyr Tyr Leu Asn Val Ile Gln Ile Ala Asp Asn Thr Gln Gln Lys
645 650 655
Leu His Glu Val Lys Arg Ile Asp Gly Asp Lys Val Val Gly Val Gln
660 665 670
Leu Ala Asp Arg Ile Val Thr Phe Ser Lys Thr Ser Glu Thr Val Asp
675 680 685
Arg Pro Phe Gly Phe Ser Val Val Gly Lys Gly Thr Phe Lys Phe Val
690 695 700
Met Thr Asp Leu Leu Pro Gly Thr Trp Gln Val Leu Lys Asp Gly Lys
705 710 715 720
Ile Leu Tyr Pro Ala Leu Ser Ala Lys Gly Asp Asp Gly Pro Leu Tyr
725 730 735
Phe Glu Gly Thr Glu Gly Thr Tyr Arg Phe Leu Arg Trp Ser His Pro
740 745 750
Gln Phe Glu Lys
755
<210> 3
<211> 756
<212> PRT
<213> Artificial sequence
<220>
<223> description of artificial sequences: artificially synthesized sequences
<400> 3
Met Gln Thr Lys Ala Asp Val Val Trp Lys Asp Val Asp Gly Val Ser
1 5 10 15
Met Pro Ile Pro Pro Lys Thr His Pro Arg Leu Tyr Leu Arg Glu Gln
20 25 30
Gln Val Pro Asp Leu Lys Asn Arg Met Asn Asp Pro Lys Leu Lys Lys
35 40 45
Val Trp Ala Asp Met Ile Lys Met Gln Glu Asp Trp Lys Pro Ala Asp
50 55 60
Ile Pro Glu Val Lys Asp Phe Arg Phe Tyr Phe Asn Ala Lys Gly Leu
65 70 75 80
Thr Val Arg Val Glu Leu Met Ala Leu Asn Tyr Leu Met Thr Lys Asp
85 90 95
Pro Lys Val Gly Arg Glu Ala Ile Thr Ser Ile Ile Asp Thr Leu Glu
100 105 110
Thr Ala Thr Phe Lys Pro Ala Gly Asp Ile Ser Arg Gly Ile Gly Leu
115 120 125
Phe Met Val Thr Gly Ala Ile Val Tyr Asp Trp Cys Tyr Asp Gln Leu
130 135 140
Lys Pro Glu Glu Lys Thr Arg Phe Val Lys Ala Phe Val Arg Leu Ala
145 150 155 160
Lys Met Leu Glu Cys Gly Tyr Pro Pro Val Lys Asp Lys Ser Ile Val
165 170 175
Gly His Ala Ser Glu Trp Met Ile Met Arg Asp Leu Leu Ser Val Gly
180 185 190
Ile Ala Ile Tyr Asp Glu Phe Pro Glu Met Tyr Asn Leu Ala Ala Gly
195 200 205
Arg Phe Phe Lys Glu His Leu Val Ala Arg Asn Trp Phe Tyr Pro Ser
210 215 220
His Asn Tyr His Gln Gly Met Ser Tyr Leu Asn Val Arg Phe Thr Asn
225 230 235 240
Asp Leu Phe Ala Leu Trp Ile Leu Asp Arg Met Gly Ala Gly Asn Val
245 250 255
Phe Asn Pro Gly Gln Ala Phe Ile Leu Tyr Asp Ala Ile Tyr Lys Arg
260 265 270
Arg Pro Asp Gly Val Ile Leu Ala Gly Gly Asp Val Asp Tyr Ser Arg
275 280 285
Lys Lys Pro Lys Tyr Tyr Thr Met Pro Ala Leu Leu Ala Gly Ser Tyr
290 295 300
Tyr Lys Asp Glu Tyr Leu Asn Tyr Glu Phe Leu Lys Asp Pro Asn Val
305 310 315 320
Glu Pro His Cys Lys Leu Phe Glu Phe Leu Trp Arg Asp Thr Gln Leu
325 330 335
Gly Ser Arg Lys Pro Asp Asp Leu Pro Leu Ser Arg Tyr Ser Gly Ser
340 345 350
Pro Phe Gly Trp Met Ile Ala Arg Thr Gly Trp Gly Pro Glu Ser Val
355 360 365
Ile Ala Glu Met Lys Val Asn Glu Tyr Ser Phe Leu Asn His Gln His
370 375 380
Gln Asp Ala Gly Ala Phe Gln Ile Tyr Tyr Lys Gly Pro Leu Ala Ile
385 390 395 400
Asp Ala Gly Ser Tyr Thr Gly Ser Ser Gly Gly Tyr Asn Ser Pro His
405 410 415
Asn Lys Asn Phe Phe Lys Arg Thr Ile Ala His Asn Ser Leu Leu Ile
420 425 430
Tyr Asp Pro Lys Glu Thr Phe Ser Ser Ser Gly Tyr Gly Gly Ser Asp
435 440 445
His Thr Asp Phe Ala Ala Asn Asp Gly Gly Gln Arg Leu Pro Gly Lys
450 455 460
Gly Trp Ile Ala Pro Arg Asp Leu Lys Glu Met Leu Ala Gly Asp Phe
465 470 475 480
Arg Thr Gly Lys Ile Leu Ala Gln Gly Phe Gly Pro Asp Asn Gln Thr
485 490 495
Pro Asp Tyr Thr Tyr Leu Lys Gly Asp Ile Thr Ala Ala Tyr Ser Ala
500 505 510
Lys Val Lys Glu Val Lys Arg Ser Phe Leu Phe Leu Asn Leu Lys Asp
515 520 525
Ala Lys Val Pro Ala Ala Met Ile Val Phe Asp Lys Val Val Ala Ser
530 535 540
Asn Pro Asp Phe Lys Lys Phe Trp Leu Leu His Ser Ile Glu Gln Pro
545 550 555 560
Glu Ile Lys Gly Asn Gln Ile Thr Ile Lys Arg Thr Lys Asn Gly Asp
565 570 575
Ser Gly Met Leu Val Asn Thr Ala Leu Leu Pro Asp Ala Ala Asn Ser
580 585 590
Asn Ile Thr Ser Ile Gly Gly Lys Gly Lys Asp Phe Trp Val Phe Gly
595 600 605
Thr Asn Tyr Thr Asn Asp Pro Lys Pro Gly Thr Asp Glu Ala Leu Glu
610 615 620
Arg Gly Glu Trp Arg Val Glu Ile Thr Pro Lys Lys Ala Ala Ala Glu
625 630 635 640
Asp Tyr Tyr Leu Asn Val Ile Gln Ile Ala Asp Asn Thr Gln Gln Lys
645 650 655
Leu His Glu Val Lys Arg Ile Asp Gly Asp Lys Val Val Gly Val Gln
660 665 670
Leu Ala Asp Arg Ile Val Thr Phe Ser Lys Thr Ser Glu Thr Val Asp
675 680 685
Arg Pro Phe Gly Phe Ser Val Val Gly Lys Gly Thr Phe Lys Phe Val
690 695 700
Met Thr Asp Leu Leu Pro Gly Thr Trp Gln Val Leu Lys Asp Gly Lys
705 710 715 720
Ile Leu Tyr Pro Ala Leu Ser Ala Lys Gly Asp Asp Gly Pro Leu Tyr
725 730 735
Phe Glu Gly Thr Glu Gly Thr Tyr Arg Phe Leu Arg Trp Ser His Pro
740 745 750
Gln Phe Glu Lys
755
<210> 4
<211> 2271
<212> DNA
<213> Artificial sequence
<220>
<223> description of artificial sequences: artificially synthesized sequences
<400> 4
atgcaaacta aggctgatgt tgtttggaaa gatgttgatg gtgtttctat gccaattcca 60
cctaagactc atcctagatt gtacttgaga gaacaacaag ttccagattt gaagaacaga 120
atgaacgatc ctaaattgaa gaaagtttgg gctgatatga ttaagatgca agaagattgg 180
aaaccagctg atattcctga ggttaaggat ttcagattct acttcaacgc taagggtttg 240
actgttagag ttgagttgat ggctttgaac tatttgatga ctaaagatcc aaaagttggt 300
agagaagcta tcacttctat catcgatact ttggagactg ctactttcaa accagctgga 360
gatatttcca gaggtattgg tttgtttatg gttactggtg ctatcgttta cgattggtgt 420
tacgatcaat tgaagcctga agagaaaact agattcgtta aggcttttgt tagattggct 480
aaaatgttgg aatgtggtta tccacctgtt aaggataaat ctattgttgg tcatgcttct 540
gagtggatga ttatgagaga tttgttgtct gttggtattg ctatctacga tgaatttcct 600
gagatgtata atttggctgc tggtagattt ttcaaggaac acttggttgc tagaaactgg 660
ttttacccat ctcataatta tcaccaagga atgtcttact tgaacgttag attcactaac 720
gatttgttcg ctttgtggat tttggataga atgggtgctg gtaacgtttt caatcctggt 780
caagctttca ttttgtacga tgctatctac aagagaagac cagatggtgc tattttggct 840
ggtggagatg ttgattattc cagaaagaag ccaaagtact atactatgcc tgctttgttg 900
gctggttctt actacaagga tgagtacttg aactacgaat ttttgaaaga tccaaacgtt 960
gaacctcact gtaaattgtt cgagtttttg tggagagata ctcaattggg ttccagaaag 1020
ccagatgatt tgcctttgtc cagatactct ggttctcctt ttggttggat gattgctaga 1080
actggttggg gtcctgagtc tgttattgct gaaatgaagg ttaacgagta ctctttcttg 1140
aaccatcaac atcaagatgc tggtgctttt caaatctact ataaaggtcc tttggctatt 1200
gatgctggtt cttacactgg ttcttctggt ggttacaact ctccacataa caagaacttt 1260
ttcaagagaa ctatcgctca caactctttg ttgatctacg atccaaagga aactttttct 1320
tcttctggtt atggtggttc tgatcatact gattttgctg ctaatgatgg tggtcaaaga 1380
ttgcctggta aaggttggat tgctcctaga gatttgaaag agatgttggc tggagatttc 1440
agaactggta aaattttggc tcaaggtttt ggtccagata accaaactcc tgattacact 1500
tatttgaagg gagatattac tgctgcttac tctgctaagg ttaaggaagt taagagatct 1560
ttcttgtttt tgaacttgaa ggatgctaaa gttccagctg ctatgatcgt tttcgataag 1620
gttgttgctt ctaaccctga tttcaagaaa ttctggttgt tgcactctat tgaacaacca 1680
gagattaaag gtaaccaaat cactattaag agaactaaaa acggagattc tggaatgttg 1740
gttaatactg ctttgttgcc tgatgctgct aactctaaca tcacttctat cggtggtaaa 1800
ggtaaagatt tctgggtttt cggtactaac tacactaacg atccaaagcc tggtactgat 1860
gaggctttgg aaagaggtga atggagagtt gaaattactc caaagaaagc tgctgctgag 1920
gattactatt tgaacgttat ccaaatcgct gataacactc aacaaaagtt gcatgaagtt 1980
aagagaattg atggagataa ggttgttggt gttcaattgg ctgatagaat cgttactttc 2040
tctaagactt ctgagactgt tgatagacct ttcggttttt ctgttgttgg taaaggtact 2100
ttcaaattcg ttatgactga tttgttgcca ggtacttggc aagttttgaa ggatggtaaa 2160
attttgtacc cagctttgtc tgctaaggga gatgatggtc ctttgtactt tgagggtact 2220
gaaggtactt atagattctt gagatggtct cacccacaat ttgaaaaata a 2271
<210> 5
<211> 2271
<212> DNA
<213> Artificial sequence
<220>
<223> description of artificial sequences: artificially synthesized sequences
<400> 5
atgcaaacta aggctgatgt tgtttggaaa gatgttgatg gtgtttctat gccaattcca 60
cctaagactc atcctagatt gtacttgaga gaacaacaag ttccagattt gaagaacaga 120
atgaacgatc ctaaattgaa gaaagtttgg gctgatatga ttaagatgca agaagattgg 180
aaaccagctg atattcctga ggttaaggat ttcagattct acttcaacgc taagggtttg 240
actgttagag ttgagttgat ggctttgaac tatttgatga ctaaagatcc aaaagttggt 300
agagaagcta tcacttctat catcgatact ttggagactg ctactttcaa accagctgga 360
gatatttcca gaggtattgg tttgtttatg gttactggtg ctatcgttta cgattggtgt 420
tacgatcaat tgaagcctga agagaaaact agattcgtta aggcttttgt tagattggct 480
aaaatgttgg aatgtggtta tccacctgtt aaggataaat ctattgttgg tcatgcttct 540
gagtggatga ttatgagaga tttgttgtct gttggtattg ctatctacga tgaatttcct 600
gagatgtata atttggctgc tggtagattt ttcaaggaac acttggttgc tagaaactgg 660
ttttacccat ctcataatta tcaccaagga atgtcttact tgaacgttag attcactaac 720
gatttgttcg ctttgtggat tttggataga atgggtgctg gtaacgtttt caatcctggt 780
caagctttca ttttgtacga tgctatctac aagagaagac cagatggtgt tattttggct 840
ggtggagatg ttgattattc cagaaagaag ccaaagtact atactatgcc tgctttgttg 900
gctggttctt actacaagga tgagtacttg aactacgaat ttttgaaaga tccaaacgtt 960
gaacctcact gtaaattgtt cgagtttttg tggagagata ctcaattggg ttccagaaag 1020
ccagatgatt tgcctttgtc cagatactct ggttctcctt ttggttggat gattgctaga 1080
actggttggg gtcctgagtc tgttattgct gaaatgaagg ttaacgagta ctctttcttg 1140
aaccatcaac atcaagatgc tggtgctttt caaatctact ataaaggtcc tttggctatt 1200
gatgctggtt cttacactgg ttcttctggt ggttacaact ctccacataa caagaacttt 1260
ttcaagagaa ctatcgctca caactctttg ttgatctacg atccaaagga aactttttct 1320
tcttctggtt atggtggttc tgatcatact gattttgctg ctaatgatgg tggtcaaaga 1380
ttgcctggta aaggttggat tgctcctaga gatttgaaag agatgttggc tggagatttc 1440
agaactggta aaattttggc tcaaggtttt ggtccagata accaaactcc tgattacact 1500
tatttgaagg gagatattac tgctgcttac tctgctaagg ttaaggaagt taagagatct 1560
ttcttgtttt tgaacttgaa ggatgctaaa gttccagctg ctatgatcgt tttcgataag 1620
gttgttgctt ctaaccctga tttcaagaaa ttctggttgt tgcactctat tgaacaacca 1680
gagattaaag gtaaccaaat cactattaag agaactaaaa acggagattc tggaatgttg 1740
gttaatactg ctttgttgcc tgatgctgct aactctaaca tcacttctat cggtggtaaa 1800
ggtaaagatt tctgggtttt cggtactaac tacactaacg atccaaagcc tggtactgat 1860
gaggctttgg aaagaggtga atggagagtt gaaattactc caaagaaagc tgctgctgag 1920
gattactatt tgaacgttat ccaaatcgct gataacactc aacaaaagtt gcatgaagtt 1980
aagagaattg atggagataa ggttgttggt gttcaattgg ctgatagaat cgttactttc 2040
tctaagactt ctgagactgt tgatagacct ttcggttttt ctgttgttgg taaaggtact 2100
ttcaaattcg ttatgactga tttgttgcca ggtacttggc aagttttgaa ggatggtaaa 2160
attttgtacc cagctttgtc tgctaaggga gatgatggtc ctttgtactt tgagggtact 2220
gaaggtactt atagattctt gagatggtct cacccacaat ttgaaaaata a 2271
Claims (10)
1. A heparinase II comprising a sequence as set forth in SEQ ID NO: 02 or SEQ ID NO: 03.
2. A nucleotide sequence encoding the heparinase II of claim 1.
3. The nucleotide sequence of claim 3, encoding the nucleotide sequence of claim 1 as set forth in SEQ ID NO: 04 or SEQ ID NO: 05, and (b).
4. A recombinant vector comprising the nucleotide sequence of claim 2 or 3.
5. The recombinant vector according to claim 4, wherein the recombinant vector is a eukaryotic recombinant expression vector.
6. The recombinant vector according to claim 5, wherein the eukaryotic cell recombinant expression vector is any one selected from pPink-HC, pPICZaA and pPICZA, preferably the eukaryotic cell recombinant expression vector is pPink-HC.
7. A host cell comprising the recombinant vector of any one of claims 4 to 6.
8. The host cell of claim 7, wherein the host cell is Pichia or Saccharomyces cerevisiae; preferably, the host cell is pichia pastoris.
9. A method for preparing heparinase II according to claim 1 comprising the steps of:
firstly synthesizing a nucleotide sequence for coding heparinase II according to claim 1 or 2, and then combining the nucleotide sequence with a eukaryotic cell recombinant expression vector to obtain a recombinant vector;
and transferring the recombinant vector into a host cell, inducing expression, and purifying to obtain the heparinase II.
10. The method of making according to claim 9, further comprising:
the purification is carried out by using a desthiobiotin purification column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210356343.6A CN114752588B (en) | 2022-04-06 | 2022-04-06 | Heparinase II |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210356343.6A CN114752588B (en) | 2022-04-06 | 2022-04-06 | Heparinase II |
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| CN114752588A true CN114752588A (en) | 2022-07-15 |
| CN114752588B CN114752588B (en) | 2023-07-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173506A (en) * | 2011-10-09 | 2013-06-26 | 清华大学 | Method for controlling production of low-molecular-weight heparin |
| CN114181927A (en) * | 2021-08-18 | 2022-03-15 | 刘颖 | Heparinase I |
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2022
- 2022-04-06 CN CN202210356343.6A patent/CN114752588B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173506A (en) * | 2011-10-09 | 2013-06-26 | 清华大学 | Method for controlling production of low-molecular-weight heparin |
| CN114181927A (en) * | 2021-08-18 | 2022-03-15 | 刘颖 | Heparinase I |
Non-Patent Citations (1)
| Title |
|---|
| SU,H.等: "heparinase II protein [Pedobacter heparinus]，GenBank: AAB18277.1，772aa linear", NCBI GENBANK, pages 1 * |
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| Publication number | Publication date |
|---|---|
| CN114752588B (en) | 2023-07-25 |
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